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THE REACTION BETWEEN AMINO-ACIDS AND 

CARBOHYDRATES AS A PROBABLE CAUSE 

OF HUMIN FORMATION 



M. L. ROXAS 



(From the Laboratory of Agbiculturax Chemistry op the 
University of Wisconsin, Madison) 



Reprinted prom 

THE JOURNAL OF BIOLOGICAL CHEMISTRY 
Vol. XXVII, No. 1, October, 1916 



c- 



Reprinted from The Jodrnal of Biological Chumihtky, Vol. XXVII, No. 1. I9I6 



THE REACTION BETWEEN AMESrO-ACIDS AND 

CARBOHYDRATES AS A PROBABLE CAUSE 

OF HUMIN FORMATION.* 

By M. L. ROXAS. 

(From the Laboratory of Agricultural Chemistry of the 
University of Wisconsin, Madison.) 

(Received for publication, July 31, 1916.) 

The studj' of the black substances obtained when proteins 
are hydrolyzed in strong acid solution is of great interest at the 
present time on account of their bearing on the natural melanins 
and on the quantitative determination of certain amino-acids 
in proteins. Grindlej^' and his coworkers state that humin 
nitrogen causes an error in the analj'sis for amino-acids of com- 
mon foodstufTs when the Van Slyke amino nitrogen determination 
is directly applied to them. This view on theoretical grounds 
was also expressed by Hart and Bentley.- It is therefore very 
important to know more about the structure and mode of forma- 
tion of these compounds. 

Mulder' was the first to show that albumins separate flooculi of a brown 
or black color on being boiled with concentrated hydrochloric or sulfuric 
acids. Hausmann-* made similar observations with globin. Samuelly" 
pointed out that the formation of these "artificial melanins" or "melanoi- 
dins" might be a secondary reaction between amino-acids and carbohy- 
drates. Maillard^ conducted experiments along this line and found a 

* The work described in this article forms part of a thesis submitted 
in partial fulfilment of the requirements for the degree of Doctor of Phil- 
osophy in the University of Wisconsin. 

' Orindley, H. S., and Slater, M. E., /. Am. Chem. Soc, 1915, xxxvii, 
2762. 

'- Hart, E. B., and Bentley, W. H., J. Biol. Chem., 1915, xxii, 477. 

3 Mulder, G. J., in Mann, G., Chemistry of the Proteids, London, 1906, 87. 

■* Hausmann, W., Z. physiol. Chem., 1900, .xxix, 140. 

' Samuelly, F., Beitr. chem. Phys. u. Path., 1902, ii, 355. 

'Maillard, L. C., Compt. rend. Acad., 1912, cliv, 66. 
71 



72 Humin Formation 

numlKT of them reacted with sugars. His experiments, however, were 
carried on in aqueous solutions at a very high concentration and tem- 
perature and it is doubtful whether under these conditions the reaction 
is similar to what takes place in the formation of either the "natural" 
or artificial melnnins. It will be shown lator in this paper that not all 
the amino-acids found reactive by Maillard reacted at all at low con- 
centration in water. Gortner and Blish' made the important discovery 
that when trj-ptophane is boiled with sugar in 22.9 per cent hydrochloric 
acid solution SO per cent of its nitrogen is converted into humin nitrogen. 
They conclude from their experiments that tryptophane alone is responsi- 
ble for humin formation. Grindley and his coworkers' disagree with this 
conclusion since they found evidence that other amino-acids give the same 
reaction. 

In view of these conflicting statements and in the hope that the 
study of the reaction between amino-acids and carbohydrates 
would throw some light on the structure and mode of formation 
of the humin substances, it was thought worth while to determine: 
(1) Which amino-acids react with carbohydrates under a given 
set of conditions. (2) Whether difi'erent sugars behave alike 
toward the same reactive amino-acid. (3) What group of the 
reactive amino-acids takes part in the reaction. 

REVIEW OF THE LITEIUTUHE. 

Udnlnszky' and Hoppe-Seyler' have shown that when sugar is boiled 
with aci<ls humin substances are formed, and if boiled in the presence of 
a nitrogenous material, the humins may also contain nitrogen. Udrdns- 
zky found, for example, that glucose and urea boiled together in strong 
hydrochloric acid solution formed humins which contained about 6.73 
per cent nitrogen. 

Saniuelly' was the first to study the behavior of humins, or "melanoi- 
dins," towards oxidizing and reducing agents. lie prepared his "melanoi- 
dins" from commercial scrum albumin according to Schmicdeberg's'" 
method modified by himself. He subjected his product to the action of 

' Gortner, R. .\., and Blish, M. J., J. Am.Chem. Soc, 1915, xxxvii, 1030. 
After this work was completed and t^ent for publication another article 
by Gortner on humin formation appeared (J. Hiol. Chcm., 1916, xxvi, 
177). In this article Gortner admits that amino-acids other than trypto- 
phane may be involved in humin formation, which is in harmony with 
the reoulls reported here. 

» Idnlnszky, L. v., Z. physiol. Chcm., 1.S&8, xii, 33. 

» IIop|)c-.Soyler, F., Z. physiol. Chem., 1SS9, xiii, 06. 
" SchmiedclxTg, O., .Arch. cxp. Path. u. Pharm., 1897, xxxix, 1. 



i\I. L. Roxas 73 

HI and PI, in a closed tube kept for S hours at 200-210°C. Among other 
products he obtained pyrrol, as detected by the color reaction with pine 
shavings, and either pyridine or piperidine or some derivative of either 
one of these bases. Ammonia was also evolved in largo amounts. None 
of the reduction products obtained as above gave indol or skatol on fusion 
with alkalies, while the humins before reduction gave on similar treatment 
an unmistakable odor of both. Samuelly also tried reduction with zinc 
dust in a current of hydrogen. From this treatment he obtained pyridine, 
pyrrol-like substances, skatol, and small amounts of an aromatic com- 
pound of the benzaldehyde series. The same investigator prepared humin 
substances from some amino-acids and glucose. He" heated for 18 hours 
a mi.xture of 10 gm. of glucose, 50 cc. water, 15 cc. concentrated HCl (sp. 
gr. 1.19), and sufficient amount of the different amino-acids so as to have 
in the solution 0.7 gm. of nitrogen. He tried ammonium chloride, urea, 
acetamide, glycocoll, aspartic acid, cystine, and tyrosine. In each case 
he found nitrogen in the melanin. No attempt was made, however, to 
determine whether the hiunin nitrogen formation was due to adsorption 
or to a definite reaction. It is interesting to note that all of the humins 
so prepared gave off pyrrol on dry distillation with zinc dust, no pyridine, 
and on fusion with alkalies only the humin prepared from tyrosine pro- 
duced an odor of indol. Nencki'- and Berdez" obtained similar results 
with the natural melanins. By alkali fusion these authors obtained from 
tumor melanin, indol, skatol, volatile fatty acids, hydrocyanic acid, suc- 
cinic acid, and other unidentified products. Pyrrol was obtained on dry 
distillation, and after heating the melanin to 300°C. for some time, upon 
addition of an alkali pyridine was detected. 

Gortner and Blish' heated pure zein, plus tryptophane, plus carbohy- 
drate in 22.86 per cent HCl, and obtained 16.5 per cent of the total nitro- 
gen of the mixture in the humin form. When tryptophane alone was 
heated with sugar in acid solution 86.56 per cent of its nitrogen was found 
in the hvunins. On the other hand, when histidine plus zein was heated 
with acid only 0.51 per cent of the total nitrogen was found in the humins. 
This amount was almost the same as that obtained when zein alone was 
heated in acid. They did not try, however, heating zein, plus histidine, 
plus sugar, in acid. Among the conclusions these authors draw from their 
experiments are the following: (1) The humin nitrogen belongs to "no 
amino-acids other than tryptophane." (2) "The reaction involved .... 
is probably the condensation of an aldehyde with the — NH group of the 
tryptophane nucleus." (3) Histidine can be eliminated "as a factor in 
the formation of humin nitrogen." 

Grindley and Slater' have tried to apply the Van Slyke amino nitrogen 
determination directly to the anal3'sis of feedingstuffs. As is to be expected, 

" Samuelly, Beiir. chem. Phys. u. Path., 1902, ii, 383. 
'- Nencki, M., and Sieber, N., Arch. exp. Path. u. Pharm., 1887, xxiv, 
17. 

" Berdez, J., and Nencki, M., Arch. exp. Path. u. Pharm., 1886, xx, 346. 



74 TTuniin Formation 

on account of the hifili carbohydrate content of these, the humin fraction 
in their nitrogen distribution is very high, varying from 3. So per cent in 
blood meal to 15.79 per cent in alfalfa hay, expressed as per cent of the total 
nitrogen. In discussing the origin of these humin substances these investi- 
gators disagree with the conclusion arrived at by Gortner and Blish, that 
the humin nitrogen of protein hydrolysis has its origin exclusively in the 
tryptophane nucleus, since they have obtained "results that clearly indi- 
cate that in addition to tryptophane a numlwr of other amino-acids when 
gently boiled with 20 per cent HCl for 24 to 30 hours in the presence of 
pure glucose give humin nitrogen. Preliminary experiments show that 
under the above treatment 4.7 to 6.3 per cent of the total nitrogen of lysine 
and cystine respectively is separated as humin nitrogen." 

Since Bourquelot and Bcrtrand discovered tyrosinase, this enzjTne has 
received much attention from a great number of investigators. Only 
that part of the work relating to the action of tyrosinase on the different 
amino-acids and related substances will be reviewed here." 

The effect of tyrosina.se on tyrosine is described by Bertrand." A solu- 
tion of tyrosine to which an extract of fyrosin.ise is added first becomes 
red, then inky black, and finally deposits a black precipitate. He proved 
definitely that atmospheric oxygen is essential to the change by conduct- 
ing experiments in vacuo and in the air. Von Fiirth and Schneider" used 
the blood (heniolymph) of the pupse of a butterfly, Dciciphila elpenor. 
They separated the enzyme from the other substances in the blood by frac- 
tional precipitation with ammonium sulfate. It was found to give a yel- 
lowish red substance with pjTochatechol ; with hydroquinone it gave a 
red solution, which then became turbid and finally deposited a consider- 
able brownish precipitate. It also acted on adrenalin, giving a dirty brown 
color. Oxyphenylethylamine became yellowish brown and finally gave 
an olive-colored precipitate. But tyrosinase has no action on casein 
itself. The same authors isolated the black substance produced from ty- 
rosine by the tyrosinase of Dciciphila puptc and determined its elemen- 
tary composition. Below is given a comparison between the percentage 
composition of this black substance and of tyrosine respectively:. 

Black lubfltanoo (humin) 

from tyranno. TjTosinc. 

perctnt p<rcenl 

C 55.66 59,60 

H 4.45 0.08 

N 13.74 7.74 

26 37 26.58 



" Bourquelot and Bertrand, G., Bull. Soc. Mycol., 1896, xii, 18. A com- 
plete list of references up to the time of its publication is found in Kastle's 
Oxidases, Hull. Hyg. Lab., 59. 

'* Bertrand, G., Compl. rend. Soc. bioL, 1896, cxxii, 1215. 

"Von Kiirth, O., and Schneider, H., Beitr. chem. Phys. u. Path., 1901, 
i. 229. 



M. L. Roxas 75 

In the formation of this "artificial melanin" from tyrosine there is an 
increase in the nitrogen content froni 7.74 to 13.74. Such an increase is 
only conceivable in one of two ways; either there is a breaking up of the 
tyrosine molecule, or some other nitrogenous substance besides tyrosine 
takes part in the formation of the melanin. The latter must be the case 
since tyrosinase, being but a weak oxidizing agent, would be unable to 
break down the benzene nucleus. The nitrogenous compound that took 
part in the reaction must evidently have come from the tyrosinase prepara- 
tion itself. This black product also yields a skatol-like odor on fusion 
with alkali. In connection with the wide distribution of tj-rosinase in 
both the vegetable and animal kingdom the following is quoted from 
Kastle's monograph : 

"Von Fiirth and Schneider are therefore of the opinion that probably 
wherever melanotic pigments occur in the living tissues of the lower and 
higher animals they originate as the result of the action of appropriate 
enzymes on substances of aromatic nature. They point out in this con- 
nection that Salkowski and Jacoby have shown independently that ty- 
rosine results from the autolysis of various animal tissues. It would seem 
likely, therefore, that in the formation of melanotic pigments two ferments 
are jointly concerned: one, an autolytic ferment capable of splitting off 
tyrosine or a similar aromatic complex from the protein molecule, and the 
other tyrosinase, which transforms the tyrosine into melanin." 

But one of the most interesting phases of the investigations on tjTosinase 
is that relating to its effect on the products of protein degradation and re- 
lated substances. Bertrand and Rosenblatt'' have found that this enzyme 
acts equally well upon racemic and Z-tj-rosine. Chodat and Staub'' dis- 
covered that albumoses do not give a red color with tyrosinase but glycyl- 
tyrosine anhydride gives such a coloration. In a later article'' these 
authors observed that glycine, leucine, and alanine retard the action 
of tjTOsinase; that dipeptides such as tyrosine anhydride, and glycyl- 
tyrosine anhydride produce yellow substances which do not become black 
as does tjTOsine itself. When, however, glycine, leucine, or alanine is 
present, a red coloration similar to that resulting from tyrosine is ob- 
tained : glycyltyrosine anh}'dride with glycine gives a rose color changing 
to bluish green; with alanine the color is deeper red, with leucine deep 
brown. But their most striking discovery is that phenylalanine is not 
acted on by tyrosinase. This, however, acts readily on p-cresol, less 
readily on jn-cresol, and still less readily on o-oresol. In fact these same 
authors observed that the enzj-me acts most readily on the para-homo- 
logues of phenol. Amino-acids like glycine increase the rapidity of the 
action of tyrosinase on jj-cresol, producing a \dolet color which ultimately 
becomes blue. Bei'trand undertook to investigate the action of tyrosinase 

" Bertrand, G., and Rosenblatt, M., Compt. rend. Soc. hiol., 1908, cxlvi, 
304. 

"Chodat, R., and Staub, Arch. Sc. Phys. Nat.; 1907, xxiii, 265; xxiv, 
172. 



76 Humin Formation 

from wheat bran on compounds analogous to tyrosine and to phenylalanine; 
that is, compounds with and without the phenolic hydroxyl group. Thus 
he found phenylalanine, phenylcthylamine, phenylmethylaniine, phenyl- 
aminoaeetic acid, phenylpropionic acid, phenylacetic acid, alanine, and 
glyeocoll produced no coloration at all. On the other hand the following 
compounds with phenolic hydroxyl groups produced coloration as follows : 

Tyrosine Grenadine-red, inky black. 

p-Hydroxj-phenylcthylamine Grenadine-red, olive-black. 

p-Hydroxyphenylmethylamine Orange-yellow, orange-red, clear ma- 
roon. 

p-Hydroxyphenylamine Orange, mahogany-red, brown. 

p-Hydroxyphenylpropionic acid Orange-yellow, grenadine-red, brown. 

p-IIydroxybcnzoic acid Rose, orange, yellow. 

p-Cresol Yellow, orange, red. 

Phenol Yellow, orange, red, brown. 

He concludes, therefore, that tyrosinase acts only on those compounds 
containing a phenolic group. 

In 1907 Abdcrhalden and Guggenheim" published an interesting arti- 
cle on the effect of tyrosinase from liiissula dclica on tyrosine, tyrosine- 
containing polypeptides and other related substances. They observed 
that glyeocoll, (/-alanine, (/-valine, /-proline, (/-serine, (/-/-isoserine, and 
/-phenylalanine retard the action of tyrosinase on tyrosine only slightly un- 
less present in very large concentrations. The largest concentration used 
was molar; /-aspartic acid and (/-glutamic acid, however, even when pres- 
ent at a concentration of 0.01 molar retard the action considerably. 
The same authors found that the enzyme has no effect on diiodotyrosine, 
/-phenylalanine, /-proline, or cystine. But /- and (/-tyrosine, homogen- 
tisic acid, and tryptophane showed a color cliango. Particularly interest- 
ing was the case of (/-tryptophane. The authors state that at first they 
thought that the coloration with tryptophane may be due to traces of 
tyrosine. They, however, used a very pure product. They repeated 
their experiment but always came to the same result. Furthermore, 
they tried the effect of tyrosinase on solutions of tryptophane-containing 
polypeptides and found development of color. They therefore conclude 
that this coloration must not be ascribed to the presence of traces of tyro- 
sine. Still more interesting is the fact that neither indol nor skatol were 
found to produce coloration. Abdcrhalden and Guggenheim in the same 
article describe the effect of tyrosinase on polypeptides containing tyro- 
sine. The color developed in these ctises is modified to some extent by 
the nature of the amino-acid combined with the tyrosine in the polypeptide. 
Addition of some .amino-acids were also found either to accelerate or to 
retard the action of tyrosinase on the polypeptide. Thus proline accoler- 

'• .'Vbderhaldcn, E., and Guggenheim, .\1., Z. physiol. Chctn., 1937-OS, 
liv, 331. 



M. L. Roxas 77 

atcs considerably the action of the oxidase on glycyl-^ty^osine anhydride, 
while aspartic acid and glutaminic acid retard the action. On the other 
hand halogen derivatives of the polypeptides were not acted upon by ty- 
rosinase. The same authors also found, as did Bertrand, that tyrosinase 
acts on phenol, giving a brown color, which was modified by amino-acids. 
Thus glycocoll plus phenol gave a cochineal color, while proline and phenol 
gave violet. The authors finally concluded that the amino-acids, when 
present, apparently take part in the production of the pigment. In a 
later article" these same authors point out that tyrosinase acts on adrena- 
lin with the rapid production of a red color and ultimately dark red floc- 
culi. All three isomers of adrenalin are affected with equal rapidity. 

It is to be regretted that in none of the above cited contributions was 
either arginine, histidine, or lysine tried. It is hoped that this omission 
will be filled in the near future. 

EXPERIMENTAL. 

The fact that zein, when boiled with glucose in 22.68 per cent 
hj'drochloric acid solution, increases its humin nitrogen from 
0.56 to 1.8-i per cent indicates, as Grindley and his coworkers^ 
suggested, that other amino-acids besides tryptophane take part 
in nitrogenous humin formation. Only a small per cent of some 
of these amino-acids may take part in this formation so that only 
by working with the individual amino-acids is it possible to de- 
termine whether the humin nitrogen was due to a definite reac- 
tion or to an adsorption. Again it is only by working under 
approximately the same set of conditions that it is possible to 
detect differences in behavior between the different amino- 
acids. The following procedure was, therefore, adhered to as 
consistently as practicable. 

The amino-acid, plus sugar, plus 50 cc. of water or hydrochloric 
acid solution of the specified strength was heated for 48 hours 
in a 300 cc. Kjeldahl flask on a sand bath. The flask was pro- 
vided with a reflux condenser made from a large test-tube fitted 
with cork and tubings for a current of cold water. After heat- 
ing, the digestion mixture was neutrahzed with the calculated 
amount of sodium hydroxide solution. The salt thus formed 
coagulated most of the precipitate that may have existed in a 
colloidal state in the solution. The mixture was then filtered into 
200 cc. graduated flasks and the humin was washed repeatedly 

'» Abderhalden and Guggenheim, Z. physiol. Chem., 1908, Ivii, 329. 



"^8 Iluniin Formation 

witli boiling water until the flask was filled to the mark. This 
amount of washing was found to be sufficient to remove almost 
all of the adsorbed amino-acid which could be removed by this 
treatment alone. The humin with the filter paper was then 
Kjeldahled. The filtrate was either Kjeldahlcd or \'an-SIyked 
or used for both determinations. 25 cc. portions were taken for 
the Kjcldahl and 10 cc. portions for the Van Slyke determination. 

The nitrogen content of the amino-acids was determined either 
by Kjeldahl's or by Van Slyke's method or by both. The per 
cent of nitrogen was the only thing used to establish the identity 
and purity of the compounds. 

The following amino-acids were furnished by Professor Hart: 

found. TheoroUosI. 

per imt pa- cml 

j^*'"°« 15.70 15 75 

^y^""® 11.2-11.0 11.67 

Ty°8''"' 7.67 7.72 

Lysine hydrobromide 

(2C,H„N,0, . HBr . H,0) 14.72 1432 

Tryptophane 6 .44 (Amino N) 6 80 

Phenylalanine* 895 g g^ 

• The phenylalanine wius kindly furniahod by Dr. T. B. Osborne of 
New Haven. 

The following amino-acids were prepared : 

^'"'■■°--''- Found. ^''"*«°Th«,«.U«.. 

IMT cent pa- oral 

Leucine, from zein 10. 90 10.70 

Proline, from zcin, also from KPlatin 12.20 12 17 

„, (No amino N) 

Cilutiuninic acid, from gliadin 7.17 75^ 

Arginine (freeHrom gelatin 29 9.5 32 ^j 

'^'."'"°-'^' 7.'70 8.04 

Histidine dihydrochloride, from blood 17 2.5 I8 f 

^'""N 5-6 g-,- 

In the preparation of the above amino-acids the directions 
given in Abderhalden's Arbeilsnwthodcn were followed. The 
per cents of nitrogen found for arginine and histidine respectively 
were not quite up to the theoretical, but since the amino nitrogen 
was almost one-fourth of the total in the arginine .sample and one- 
third in the histidine, it was evident that the samples of both 



M. L. Roxas 79 

these amino-acids were free from other amino-acids, their low 
total nitrogen content being due to moisture. The nitrogen 
determinations of these amino-acids were made on the same day 
that the experiments on humin formation were started. 

Due to the scarcity of material it was found impossible to 
recrystallize some of the amino-acids in order to obtain as pure 
a product as could be desired. 

The results are shown in the following table. All the experi- 
ments were in duplicate and average figures are given. 

The results show that neither alanine nor leucine give humin 
nitrogen. Glutaminic acid when boiled with sugar in 2 per cent 
acid solution yields some humin nitrogen, but none in 20 per 
cent acid. Attention is called to the fact that glutammic acid 
on heating even at the concentration used seems to form pyrro- 
lidon carboxyHc acid readily, as evidenced by the loss of activity 
of its amino nitrogen in Experiments 13, 14, and 16. Such a 
formation does not take place in strong acid. Phenylalanine 
yields about 1.65 per cent of its nitrogen in the humin in 20 per 
cent acid solution. Proline does not give humin nitrogen with 
glucose with 20 per cent acid, but seems to react to some extent 
with xylose and fructose in 4.15 per cent acid solution. Cj^stine 
with 20 per cent HCl jdelds about 3.1 per cent humm nitrogen 
and the noteworthy fact about this amino-acid is the deeply 
colored filtrate it produced. The same was observed with the 
filtrate from the tyrosine-sugar experiments. As much as 15 
per cent of tyrosine nitrogen may be converted into humin nitro- 
gen. 

The cases of the three hexone bases are particularly in- 
teresting. When boiled with sugars in 20 per cent HCl solution 
all three yield some of their nitrogen as humin nitrogen. Ar- 
ginine and lysine, with sugar, give more deeply colored filtrates 
than histidine. If the deep coloration of the filtrate indicates 
reaction, then it must be stated definitely that in the case of 
cystine, tyrosine, arginine, and lysine in 20 per cent HCl, the hu- 
min nitrogen is due to a reaction and not to an adsorption. An- 
other fact that supports the contention that a definite reaction 
is responsible for humin formation at least in the case of tyrosine 
is that phenylalanine gives but little humin nitrogen. If this were 
a case of adsorption, then there should probably be no differ- 



80 



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M< 


00 


(M 


■* 


,_, 


-* 


(^ 


■ OD 


oo 






c^ 


fM 




'-' 




o-l 


C-) 


(M 





CO , o o 

Tf 3 o o 

o ~ o o 



+ : + 



o W 
+ : + e 



2 3 



^ IM Q 



a w a 



to 5 

2 & 



o : 

csi ri 

d H 

+ c 



2 S 2 



r« 


^ 




d 


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>. 


!-i 






f^n 










j:5 


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, , 




+ 





+ § S « 



THE JOURX.VL OF BIOLOGICAL CHEMISTRY, VOL. XXVII, NO. I 



82 



Huniin Formation 









Urn 




















_o 
















"o 


ei 














V 


_o 


« 


2 


: 








■^ 


"o 










.a 




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>. 










a 




a 


.s 


, 


, 


, 




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c: 


u 


■ 


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" 




& 




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, "~ 














2 S 


J 


2 


2 


- 








^ CJ 
































b 












- I^^ll 




8 


8 


8 


8 






H g.a 








'^ 








e 

ToUl 
N 

found 
(col- 
umn 1 
-(-col- 
umn 2). 




S-: 


<j « 


'^ ^ 3»0 *» M*=^ *^ 






t 


S§ 


s.i 2 


O^C ••t-iO -Nl-C 

2S.8 2SS.S slag 






S is 


E 














< -o" 
















3-5a -iei 
















O-^'a .5- 














tj 


O 1 -^ 














1 


° S2 oil 














'.a 


< — - _«, 














§ 








CN 


o 


00 




~ Jz'tlill 




O 






o> 




o 


E 


•^ 


-r 


n 


CO 




J. 


H .SS=.o 














s 
























a 


a 




? 


1 

Hurain N 
(Kjddnhl). 


1 


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d 


s 

o 




2| 


8 

d 










+ 


+ 




+ 




8 ': 










g 


S 




s 




+ : 











o 
u 
_3 


o 




3 




§ : 










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u 








c 












3 • 




B 






i 


1 








•a : 




1 




s i. 


s^ 


8 C 


s S 


i i 




1 




CLi " ■ 


o ._ 

c 


is 

, a 


fix 

° c 


o 








-f 


+ £ 


+ 8 


+ 8 


+ w 










, 1. 


_: *■ 


i- 


•^ 








C 


E ^ 


IS. 


1^ 


ii 












lO 


1(5 










•c 


•c ^ 


115 ^ 


■ .■5 — 










~ 


M T 


ZI -fl" 


Z^ I- 


S o. 








6 


d 


d 


d 


d 




|P 




8 


?i 


S 




S5 




s 



M. L. Roxas 



83 






00 








CO 








« 


g 




^ 








^ 








M 


t^ 

-:(< 




g 
^ 


■^ 

g 







3: 


CO 
05 


t-. 


g 


CO lO 

d> ci 

CS) CO 


per 
cent 
47,31 
99.4 


S S 
0. u 


to 








2 














o 

1 









1 














5: 








5 














CO 








CO 























s 














^ 








CO 























(M 








00 


g 




^ 








CO 



















.s s 

CO 

Ocdo 






o — 



rM 





+ 


K 






Fi 


c 

ID 


bD 






3W 






o + 
+ 9 



84 



Iluinin Format ion 



?5 S 






1^ 00 5; 









=z"e55 



c. sS-s4 



o c o 






OJ — O M o 

N o -r tc o 

£J si d 2 ^ 



s-£ 



o o — 



0-2 



O 



+ K 






a . ^ ^ 



+ c + 

-r ci 



-^ ^ O 



s 


s 


+ 


08 


u 


+ 


c 
o 

2 


3 








ec 


O 








M. L. Roxas 



85 



o 

CI 




§ 








g 








8 


§ 


o 

o 


s 


s 




00 








C5 










C<1 




(M 




a o 
ft o 










00 


o 


p. 


G ■ 










8 




o 








§ 










o 


5S 


S- 


o 




o 








■* 










-^ 


o 


'^ 


o 
to 




o 








§ 








o 

o 


o 

00 




s 














g 








Tt< 


cc 


CO 


CO 


o 




o 
o 








o 








o 
o 


o= 


00 


CO 














s 








^ 


(M 


!M 


c^ 


o 




^ 








CO 




























00 

















" =^ c 

+ K 






K "Si 



ago 
aco M 
° o ^ 

+ + g 

a s o 

bO bC p. 



s 

6J0 
O 

Kg 

OK 



lO ft 



o o 

+ "^ 



a a 



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86 



Ilumin Formation 











c 






































it 

c 






i 






s 




: 


o 

k- 








o 




i 






Q 






.Ml 






. 




. 


a 

o 

T3 








S 

B 




£ 






























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c 


























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5 






: 




- 


- 








- .S 


















































































_fc 




























h- 


Hi 


E 




c 

■« 






O 






c 




■>f 


P5 








t?5 




Total 
N 

found 
(col- 
umn 1 
-(-col- 
umn 2). 


Ck 










1^ 


* fc 


c 


1 


r u 


^^°..^ 


»C 


CD t. 


c 




o< X |_ -g 




E 










s 


o a 


ZJ 


g 


§ a 


sisg as 


si 


s s. 


g 




2§ S.S 




.S ^8 






s 






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1 




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^ 








o 


A 


ezs| 


1 




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^- 




d 


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d 




< -3 = 












1 






1 














1 




S^J^as 


Ck 




^ 






g 






§ 




s 


o 








s 




U-^a -- 


E 




« 






gi 






g 




- 


^ 








M 










5 






s" 










s 


g 








K 




(» 




































E 










1^ 






S 




= 


00 








w 


" 


Total 

trat« 
(Kiol- 
dahl). 


E 
















1 
g 




{3 


53 








5 




































.•* 












c 






a 






s 






B 






a 




:ir^ 








o 






o 






c 






c 






» 




2 














CJ 






c 






&.< 






o 




■§■5 


i 




i 


I 




s 


u 








8 


r* 


s 






Sg 6 




i^ 


E 




c 


a 




d 


B. 




c 


Q. 


d 


d 






W O- 




i'6 








Cv| 






CO 






w 






£i 






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CS 


















so 






















































^^ 






^H 






^^ 






d 






1^ 








+ 






+ 






+ 






i 


S 






8 

■f 












S" 












3 






+ 

S 


+ 

O 

3 




c 


3 








C 

1 




d 






U 

o 






q 






1 


1 




c 
C. 


i 

q 








H 




+ 






(N 


^ 




ci 


o 




O 


q 








u 












-f- 


hH 




+ 






+ 


+ 




H 


+ 


K 








i 


i 
1 




8 


c 

& 




i 

8 


c 
c 




1 








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d 






d 






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s 


"5 






S 







M. L. Roxas 87 

ence in behavior between tyrosine and phenylalanine. In aque- 
ous or very weak acid solution arginine, histidine, and lysine 
evidently react with sugar as indicated b}' the highly colored 
solutions produced and by the loss of activity of a large fraction 
of their amino nitrogen. Thus, when arginine plus glucose 
was boiled in water there was a very deep coloration of the solu- 
tion (Experiment 39), and at least 25 per cent of the amino nitro- 
gen became inactive towards nitrous acid. Lj^sine behaved 
similarly (Experiment 42), 17 per cent of the amino nitrogen 
becoming inactive towards nitrous acid. Histidine acted like- 
wise (Experiments 47 and 51), 16.2 per cent of its amino nitro- 
gen becoming inactive. These facts show that in the cases of 
histidine and arginine the a-amino nitrogen takes part in the 
reaction. In the case of lysine it is difficult to establish which 
amino group is reactive, since at the time the amino nitrogen in 
the filtrate was determined the temperature in the laboratory 
was about 35°C. and at this temperature it was found that both 
the a- and the e-amino group of lysine react with nitrous acid in 
5 minutes, as may be seen in the amino nitrogen determination 
of the filtrate (Experiments 40 to 42). It is to be noted that in 
these cases some loss of nitrogen also took place. It may be 
that during the reaction some ammonia was given off. 

The result with tryptophane is in agreement with the work of 
Gortner and Blish' in that a greater portion of the tryptophane 
nitrogen is converted into humin. The strength of the acid used 
here and the different procedure followed may account for the 
difference in the per cent of tryptophane nitrogen foimd in the 
humin which according to the above named authors was 86 per 
cent while in these experiments only about 71 per cent was ob- 
tained. Due to a lack of material it was impossible to repeat the 
experiment with tryptophane. 

In order to determine which atomic groupings in tyrosine, 
cystine, and tryptophane were responsible for humin formation, 
the humin from each one of these amino-acids was dissolved 
in 0. 1 N alkali and Van-Slyked. It was believed that if the amino 
groups . in this humin remained mtact they should still give the 
nitrous acid reaction. The results are as follows: 



88 Hiimin Formation 

Humin nitroffon. Reactive with HNO9. 
mg. nif/. 

Tyrosiiu" 2.360 2.4>5 

Cystine 0.974 0.88 

Tryptophane 13 820 190 

I''ioni these results it must be concluded that in the case of 
tj'ioisine and cystine it was not the amino group that reacted 
with sugar to form humin but some other group, probably the 
(OH) in tyrosine and the (S-S) in the case of the cystine. If this 
were the case, then the cystine would presumably undergo re- 
duction before reacting with the sugar. 

In order to determine whether, as Gortner and Blish suggested, 
the furfurol obtained from sugar was responsible for the reaction. 
Experiments 54, 55, and 56 were performed as follows: 

Exporinieiit Humin N. Poroeotof 

No. m). tolal N. 

54 0.2 gni. cystine + 2 cc. furfurol + 20 

per cent HCl 7.00 32.0 

.•i.'i 0.2 gin. tyrosine + 2 oc. furfurol + 20 

per cent HCl 8 40 55.0 

.">fi 0.2 gin. nrginine + 2 cc. furfurol + 20 

percent HCl.. 12 7.5 21.5 

These results tend to show that the furfurol formed from sugars 
under the influence of acids may to a great extent be responsible 
for humin formation. 

.\s to the effect of the different sugars on the reactive aniino- 
acid. Kxperiments 20, 21, 22, 29, 38, and 46 show that xylose 
ant! fructose give higher results than glucose as a rule. This is 
to In- expected, if it is admitted that furfurol or some other sim- 
ple aldehyde is the active substance in these reactions. 

DISCUSSION OF RESULTS. 

Some evidence is given which shows that the a-amino groups of 
argminc, histidine, and tryptophane take part in the reaction 
with sugars. On the other hand, the a-amino groups of alanine 
and leucine arc unai>lc to give the same reaction. Glutaminic 
acid and [ihenylalanine, although giving some humin nitrogen, 
Ukewisc furnish no indication of reaction. At least for the pres- 
ent it may be admitted that the humin nitrogen in these cases — 



M. L. Roxas 89 

glutaminic acid and phenylalanine — was due to adsorption and 
not to a reaction. It was also shgwn that in tj'rosine the reactive 
group is presumably the (OH) and surely not the a-NHs. In 
cystine, as shown above, the a-amino group remained intact, 
so that presumably the reaction was with the mercaptan group. 
The question may then be asked: Why are the a-NH^ groups of 
arginine, histidine and tryptophane more reactive than those 
of the other amino-acids? An attempt to explain this difference 
in behavior of the amiuo-acids towards carbohydrates, based 
on the present work and on some of the contributions reviewed 
in the first part of this paper, is here offered. It is generally 
stated that the properties of a compound are functions of its 
structure. It is, therefore, to the structure of these amino-acids 
that we must look for an explanation of then- different behavior 
towards carbohydrates. The structural formulas of histidine, 
tryptophane, and arginine are given below. 



CH 


NHz 


/-\ 


/ 


N N— H(b) (c) 


NH=C 


H^N 


\(f) 


1 


NH-CH2 


[-C = C— CHo— CH 


\ 


(a) 1 


( 


COOH 


/ 


Histidine. 


NHa— CH— CHj 




(h) \ 




COOH 




Arginine. 



CHo 




CH-COOH 



NH 

(d) 

Tryptophane. 

Several investigators have advanced the idea that humin 
formation is dependent on the presence of labile hydrogen in 
the amino-acid molecule (SamueUy, Grindley and Slater, etc.). 
Evidently judging from the results of the present work the two 
hydrogens of the a-amino groups of alanine and leucine are not 



90 



Ilumin Formation 



labile enough to give condensation products with carbohydrates 
at least under the conditions of these experiments. 

In histidine, arginine, and tryptophane, however, there are 
other labile hydrogens (a, b, c, d, c, f). The positions of these 
labile hydrogens with respect to the a-amino group are very favor- 
able for ring formation. The reaction with a carbohydrate 
or finfurol may very well be thought of as taking place as follows: 



Histidine. 



CH 



H 

I 
C-R 

4- 



/CH; 



■Xc/" 



N N'^0 h;.NH 



.NH— C-^ v.. 

CH II |\x)OH 



HC=Cy /C 

^ch/ 



< 



COOH 



CJI O H;NH 



/\ 



NfH \ / x:ooH 




/H 



H 



_^(d.i};nh 



I 

R 
III 



|^CXX)H 

I 



X/X/oJi o h;nh 

NH --il-' 



IV 



Arginine 
NH,v/N— CH, — CH, 

J-- ' 

^ ill \ /CH, 

tiw^ >oh;nhc< 

H— C COOH 



M. L. Roxas 91 

The following facts tend to support the idea of ring formation : 

1. The intense color of the products. 

2. Miss Homer-' in her work on the condensation products 
of tryptophane with aldehydes, speaking of the action of glyoxal 
on this amino-acid, states: 

"Taking into consideration the necessity of the presence of an oxidiz- 
ing agent and also the fact that the substance produced is intensely colored 
it is highly probable that in this reaction, besides the simple aldehyde 
condensation .... there has also been elimination of hydrogen 
accompanied by complex ring formation." 

3. The fact that pyridine was obtained by Samuelly from his 
"melanoidins," was at one time used as an argument to indicate 
that a pj'ridine nucleus was found in proteins. This idea has 
been disposed of by Emil Fischer's work on proteins, but the fact 
remains that pyridine is found in the humin formed from pro- 
teins. This occurrence may be explained by Reactions II and IV 
thus; 

yCHU y „ I /Cnd , „ 



4. The action of tjTosinase on tyrosine tends to support the 
idea of ring formation. Tyrosinase produces coloration with 
tryptophane but not with indol, skatol, or glycocoU. There- 
fore, the formation of the highl}' colored product requires the 
peculiar structure of tryptophane. This formation may be con- 
sidered as taking place in the manner described above. 

The differences in behavior between histidine, arginine, and 
tryptophane may again be referred back to the differences in 
theii- structure. Tryptophane being already a complex com- 
pound with a benzene and a pyrrol ring may form an insoluble 
four-ringed compound with furfurol, which is extremely resist- 

-' Homer, A., Biochem. J., 1913, vii, 111. 



92 Iliiiiiiii Forinatiftii 

ant to the action of acid. This will explain why tr.vptophane 
is converted into huniin almost (|uaiitit;itiv<ly. (hi the other 
hanil furfurol may form with hi.stidiiie and arjj;iiiinu products 
which are still more or less soluble and in the presence of strong 
acid may be hydrolyzeil back to the free amino-acids. Thus 
as in the case of Rlutaminic acid, no formation of a ring com- 
pound takes place in strong hydrocidoric acid solution. This 
view will explain why in weak acid or in aqueous solution both 
histidine and arginine react more readily to form colored products 
than in strong acid solution. 

It is not claimed that the reaction given above gives the actual 
structure of the melanin molecule, since no evidence is available 
to indicate what happens to the rest of the molecule of the amino- 
acids during the reaction with sugars. This theory on humin 
formation is given here in the hope that it may serve as a guide 
for future work on the structure of these compounds. 

No evidence was found in the present work to explain Samu- 
elly's finding that when the huniin obtained from sugar plus 
tjTOsine was fused with alkalies, an odor of indol was obtained. 
It might have been possible that the tyrosine used contained 
traces of tryjitophane which would explain the production of 
indol. Likewise the fact that pyrrol was obtained from his 
"mclanoidins" can be traced back to tiie presence of the trypto- 
phane nucleus in them. 

Almost all of the experiments recorded in this paper were done 
with single amino-acids. There was found evidence (Experiment 
31) to show that the reaction would be different, at least in the 
case of cystine and tyrosine, if other amino-acids were present 
in the reaction mixture with sugar. If cystine and proline were 
boiled together in the presence of glucose and 20 per cent HCl 
a larRcr amount of cystine nitrogen disappeared in humin forma- 
tion than when cystine was boiled alone. The same was true 
when tyrosine and proline were boiled together. It would, 
therefore, be interesting to study the behavior of mixtures of 
ilifferent amino-acids when boiled with sugars, both in acid 
and aqueous solutions. .Vbderhaldcn and Guggenheim'" work- 
ing with tyrosinase along this same line already concluded that 
other amino-acids, when present, apparently take pari in the 
jiroduction of the pigment. 



M. L. Roxas 93 



CONCLUSIONS. 



1. Alanine, leucine, phenylalanine, and glutaminic acid may be 
eliminated as important factors in humin formation, when sub- 
jected to the treatment used in these experiments. Proline, 
however, under certain condition.s may b!> involved in humin 
formation. 

2. The following amino-acids were responsible for humin 
formation, and in digestions, with 20 per cent HCl plus sugar, 
the proportion of their nitrogen disappearing was: Tyrosine, 15.0; 
cystine, 3.1; arginine, 2.33; lysine, 2.62; histidine, 1.84; trypto- 
phane, 71.0 per cent. 

3. Xylose and fructose were as a rule more reactive than 
glucose. 

4. Arginine, histidine, and lysine reacted with sugars more 
readily in weak acid or aqueous, than in strong acid solutions. 

5. Arginine, histidine, and tryptophane reacted with loss in 
reactivity of their amino nitrogen towards nitrous acid, but 
tyrosine and cystine reacted without any such loss. 

6. A possible mode of reaction is suggested. 

It is with pleasure that the writer acknowledges his obligation 
to Professor E. B. Hart, Chief of this Department, for giving 
him this problem, and for his many valuable suggestions during 
its execution. 



LlbKHKT Ul- i_unon.t->-. 



002 672 082 5 



THE VWAVtRLY PRESS 
•ALTIMOna. u. •■ *. 



